Antimicrobial low-density polyethylene (LDPE) and poly(lactide) (PLA) films were prepared by blending with grapefruit seed extract (GSE) incorporated thermoplasticized starch (TPS) using extrusion blowing and extrusion casting methods, respectively. The effect of polymer matrix was studied by comparing the film properties such as optical, mechanical, water vapor barrier, and antimicrobial properties. Scanning electron micrograph (SEM) result indicated that the TPS was homogeneously dispersed through both polymer matrix. Though transparency, mechanical, and gas barrier properties of the blend films decreased compared with their neat films, the blend films showed UV-light screening function and antimicrobial activity against food-borne pathogenic bacteria, E. coli and Listeria monocytogenes, which was mainly attributed to the GSE. While PLA/TPS film exhibited strong antibacterial activity against both bacteria, LDPE/TPS film showed lower antibacterial activity. This was presumably because the GSE was entrapped more tightly by the LDPE than the PLA. Packaging application test using minced fish paste also showed that the PLA/TPS film was more effective to prevent bacterial growth than LDPE/TPS film.